A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells
Key findings
The pcD vector combined the SV40 early promoter with two late region introns (16S and 19S splicing sequences) positioned upstream of a cDNA cloning site, plus an SV40 late polyadenylation signal downstream. This arrangement enabled cDNA inserts to undergo transcription initiation, intron splicing, and 3' polyadenylation in mammalian cells while retaining compatibility with E. coli cloning via pBR322 origin and ampicillin resistance marker.
Transfection of pcD-α-globin into COS cells produced α-globin transcripts that initiated correctly at the SV40 promoter, spliced at either of the two introns, and polyadenylated either at the globin-encoded signal or the distal SV40 polyadenylation site. RNA mapping confirmed proper transcript processing for the inserted cDNA, demonstrating the vector's ability to generate fully processed mRNAs from cDNA sequences lacking native regulatory elements.
A cDNA library constructed with mRNA from SV40-transformed human fibroblasts yielded approximately 1.4 × 10^6 clones, including full-length cDNA sequences encoding functional proteins. The library successfully identified and expressed hypoxanthine-guanine phosphoribosyltransferase (HPRT), validating the approach for isolating rare cDNAs based on protein function rather than hybridization screening.